Reversible covalent modification of proteins has been shown to be a key feature in the regulation of cellular processes. A novel type of such modification is catalyzed by enzymes which methylate carboxyl groups on proteins. These enzymes appear to be ubiquitous in eucaryotic tissues, are particularly active in mammalian brain, heart, lung, erythrocyte, and liver, but their physiological role has not been established. The methyl ester formed, however, is metabolically unstable and methylation and demethylation reactions of this type are thus likely to be involved in the control of the function of these cells. I propose to identify the endogenous carboxyl methylated proteins initially in human red blood cells and rat hepatocytes, focusing on those proteins found in the membrane fraction of the cell. Methylated species, labelled by in vivo and in vitro incubation with (3H-methyl) methionine and (3H-methyl) S-adenosyl methionine, will be identified after separation on dodecyl sulfate gel electrophoresis. The specific methylases and demethylases for these species will be purified and characterized, and experiments will be performed to assess how methylation can regulate erythrocyte metabolism by examining the consequences of methylase inhibition by permeable compounds such as S-tubericidinyl homocysteine. I will be especially concerned with the effect of methylation on membrane processes such as chloride and glucose transport, cellular shape control, and the transfer of information between plasma and cell. Finally, I propose to characterize the structure of the methylated proteins from erythrocyte and liver, determining the topography of membrane proteins by labelling experiments with permeable and impermeable reagents and determining the size, shape, and detergent interactions of these proteins solubilized in the mild detergent Triton X-100.